Time-resolved DNA release from an O-antigen-specific Salmonella bacteriophage with a contractile tail.

Myoviruses, bacteriophages with T4-like architecture, must contract their tails prior to DNA release. However, quantitative kinetic data on myovirus particle opening are lacking, although they are promising tools in bacteriophage-based antimicrobial strategies directed against Gram-negative hosts. For the first time, we show time-resolved DNA ejection from a bacteriophage with a contractile tail, the multi-O-antigen-specific Salmonella myovirus Det7. DNA release from Det7 was triggered by lipopolysaccharide (LPS) O-antigen receptors and notably slower than in noncontractile-tailed siphoviruses. Det7 showed two individual kinetic steps for tail contraction and particle opening. Our in vitro studies showed that highly specialized tailspike proteins (TSPs) are necessary to attach the particle to LPS. A P22-like TSP confers specificity for the Salmonella Typhimurium O-antigen. Moreover, crystal structure analysis at 1.63 Šresolution confirmed that Det7 recognized the Salmonella Anatum O-antigen via an ϵ15-like TSP, DettilonTSP. DNA ejection triggered by LPS from either host showed similar velocities, so particle opening is thus a process independent of O-antigen composition and the recognizing TSP. In Det7, at permissive temperatures TSPs mediate O-antigen cleavage and couple cell surface binding with DNA ejection, but no irreversible adsorption occurred at low temperatures. This finding was in contrast to short-tailed Salmonella podoviruses, illustrating that tailed phages use common particle-opening mechanisms but have specialized into different infection niches.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Evaluation of the natural virucidal activity of teas for use in the phage amplification assay.

Many natural products have intrinsic antimicrobial activity. In this study we have examined infusions from nine types of loose-leaf tea for their ability to inactivate bacteriophage, for use as an alternative to plant extract in a phage-based Salmonella detection assay. The results demonstrated that tea infusions, either freshly prepared or stored at 4 degrees C had virucidal action against two phages, Felix 01 and P22. Crucially, for use in the detection assay, there was no antibacterial effect of the virucide on the target bacteria. Therefore, tea was a good candidate to replace pomegranate as the virucidal agent in the phage amplification assay.

Regional, seasonal, and antimicrobial resistance distributions of salmonella typhimurium in Canada: a multi-provincial study.

BACKGROUND: This study was conducted to describe the geographical and seasonal distributions of reported human Salmonella Typhimurium (ST) definitive type 104 (DT104) cases, to compare these characteristics to those of non-DT1 04 cases, and to investigate specific antimicrobial resistance (AMR) patterns in four Canadian provinces. METHODS: All laboratory-confirmed ST cases originating from passive reporting in Alberta, British Columbia, and Saskatchewan, and every second case in Ontario identified from December 1999 through November 2000 were investigated. RESULTS: A total of 470 human Salmonella Typhimurium cases were identified during the study period. DT104 was the most common phage type, although its incidence varied by province. The proportion of DT104 cases living in urban Ontario, British Columbia and Saskatchewan did not differ from the general population, but in Alberta, the DT104 cases were more likely to live in rural areas. Overall, DT104 isolates were more often R-type ACSSuT compared to non-DT104 cases, and R-type AKSSuT was often associated with DT208. DT104 cases displayed no seasonality whereas non-DT104 cases were more frequent in the summer than in the winter. INTERPRETATION: Our results suggest that DT104 and non-DT104 cases vary by province, urban vs. rural residential status and by resistance patterns. Lack of seasonality in the DT104 cases may indicate a lesser influence of the agro-environmental route (i.e., farm -manure - water and direct contact) compared to the agro-food route (i.e., farm - animals -food) for these infections. Strain characterization and integration of surveillance information related to ST from animal, food and humans is warranted.

Mutagenic response of Ames strains cured of their inducible Fels 1 and Fels 2 prophages.

Ames strain TA100 was cured of its Fels 1 and Fels 2 prophages to yield the corresponding nonlysogenic derivative designated TAQ100. The two monolysogenic strains corresponding to TA100 lysogenic for Fels 1 (TAQ100F1) and for Fels 2 (TAQ100F2) were also isolated. In addition, the equivalent strains lacking pKM101 and designated TAQ1535, TAQ1535F1, and TAQ1535F2 were obtained. Ames strains TA98 and TA1538 are lysogenic for Fels 2 and were observed by colony hybridization to contain cryptic Fels 1 DNA sequences. Strains corresponding to TA98 and TA1538 cured of Fels 2 were isolated and designated TAQ98F1d and TAQ1538F1d, respectively. Fels 1 grew poorly on Fels 1-cured strains, and Fels 2 grew not at all on Fels 2-cured strains. The cured strains had therefore to be identified as such by their failure to react in colony hybridization with 32P-labeled probes of Fels 1 and/or Fels 2 DNA. The specificity of the labeled probes was confirmed with the aid of the nonlysogenic Salmonella typhimurium strain Q1 and its two monolysogenic derivatives Q1 (Fels 1) and Q1 (Fels 2). The cured strains were found to respond in the same manner as did the standard Ames strains to a variety of well-known mutagens, including aflatoxin B1, 7, 12-dimethylbenz(a)anthracene, daunorubicin, 2-amino-dipyrido[1,2-a:3',2'-d]imidazole, and beta-naphthylamine. Also, mitomycin C, bleomycin, and diethylstilbestrol were nonmutagenic to TAQ100 and TAQ98F1d as they are to TA100 and TA98. Since the Fels prophages are inducible by aflatoxin B1, by daunorubicin, and by other agents, it seems that mutagenesis and Fels prophage induction occur in separate subpopulations of cells; this situation had previously been reported to occur for mutagenesis and prophage lambda induction in Escherichia coli. In any case, the Fels prophages appear to have no major influence on the mutagenic response of the Ames strains.

DNA injection proteins are targets of acridine-sensitized photoinactivation of bacteriophage P22.

Viruses and other nucleoprotein complexes are inactivated on exposure to white light in the presence of acridine and related dyes. The mechanism is thought to involve generation of singlet oxygen or related species, but the actual molecular targets of the inactivating event have not been well defined. We have re-examined the mechanism of dye-sensitized photoinactivation taking advantage of the well characterized bacteriophage P22. Though the inactivated phage absorb to their host cells, the cells are not killed and genetic markers cannot be rescued from the inactivated phage. These observations indicate that the chromosome is not injected into the host cell. However, the DNA of the damaged particles shows no evidence of double-stranded breaks or crosslinking. The DNA injection process of P22 requires three particle-associated proteins, the products of genes 7, 16 and 20. Gp16, which can act in trans during injection, is inactivated in the killed particles. Sodium dodecyl sulfate/polyacrylamide gel analysis reveals that gp16, gp7 and gp20 are progressively covalently damaged during photoinactivation. However, this damage does not occur in particles lacking DNA, indicating that it is DNA-mediated. Similar findings were obtained with acridine orange, acridine yellow, proflavin and acriflavin. These results indicate that the actual targets for inactivation are the DNA injection proteins, and that the lethal events represent absorption of photons by acridine molecules stacked in a region of DNA closely associated with the injection proteins.

Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against gamma-irradiation inactivation of bacteriophages T4 and P22.

Bacteriophages P22, T4+, and T4os (osmotic shock-resistant mutant with altered capsids) were diluted in 0.85% NaCl and exposed to gamma irradiation (2.79 Gy/min) at room temperature (24 degrees C). T4+ was more sensitive to inactivation than was P22, and the T4os mutant was even more sensitive than T4+. Catalase exhibited a strong protective effect and superoxide dismutase a weaker protection, indicating that H2O2 or some product derived therefrom was predominant in causing inactivation of plaque formation. Low but significant (0.1-0.3 mM) reduced glutathione (GSH) enhanced phage inactivation, but a higher (1 mM) GSH concentration protected. A similar effect was found for the polyamine, spermidine. In contrast, 0.1 mM L-ergothioneine (2-thiol-L-histidine betaine) exhibited strong protection and 1 mM afforded essentially complete protection. L-Ergothioneine is present in millimolar concentrations in some fungi and is conserved up to millimolar concentrations in critical tissues when consumed by man. L-Histidine and two histidine-containing dipeptides, carnosine and anserine, protected at a concentration of 1 mM, a level at which they are present in striated muscles of various animals.

On the different response of Salmonella typhimurium hisG46 and TA1530 to mutagenic action of base analogues.

Various mutagens are known to induce more his+ revertants in TA1530 than in hisG46 strain. To test whether the mutator effect shown by TA1530 is limited to the his mutation, the lysA8 marker was introduced into both the TA1530 and hisG46 strain, and its reversibility, after induction by N4-hydroxycytidine, was estimated. The ability to reverse the lys marker was tenfold higher in the TA1530/lysA8 transductants than in the hisG46/lysA8 transductants or in the donor for lys, the lysA8 strain.

Curing and induction of the Fels 1 and Fels 2 prophages in the Ames mutagen tester strains of Salmonella typhimurium.

A method is described for curing the Ames Salmonella mutagen tester strains of their Fels 1 and Fels 2 prophages with the aid of the antitumor drug daunorubicin. Non-lysogenic derivatives corresponding to TA100 and TA1535 were isolated and designated TAQ100 and TAQ1535 respectively. In addition, the Fels 1 monolysogens TAQ100F1 and TAQ1535F1, as well as the Fels 2 monolysogens TAQ100F2 and TAQ1535F2, were obtained. Finally, strains corresponding to TA98 and TA1538 cured of Fels 2, but retaining a cryptic Fels 1 (F1d) prophage were isolated and designated TAQ98F1d and TAQ1538F1d respectively. The various cured derivatives were identified by colony hybridization with 32P-labeled probes of Fels 1 and Fels 2 DNA. Southern blot hybridizations confirmed that phage-specific Fels DNA sequences were missing from the cured strains. The Fels 2-cured strains were resistant to Fels 2, but Fels 1 grew, albeit poorly, on the Fels 1-cured strains. Strains TAQ100F1, TAQ1535F1, TAQ100F2 and TAQ1535F2 were used in prophage induction assays, in the presence of rat-liver extract where necessary. Daunorubicin, bleomycin, mitomycin C, aflatoxin B1, 2-amino-dipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) were found to induce Fels 1 and/or Fels 2 in at least one of these strains. The induction of the Fels prophages in the TAQ monolysogens may provide a useful complement to the Ames test for the detection of DNA-damaging agents and potential carcinogens.

Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of constriction of the required strains.

A 'toxicity' test protocol is described here to be used for determining the bactericidal effect of the chemicals which are tested for their mutagenic activity by the Ames method. Two sets of strains, isogenic with the Ames tester strains except for their his character, are constructed. One set is the his+ derivatives of the tester strains which are used for measuring the survival of the inoculum cells after exposure to the chemical. The other set is the stable his- derivatives of the tester strains which are used for simulating the background growth in the Ames mutagenicity plate test. The per cent survival of the his+ cells in the inoculum in the presence of the 'filler cells' is used as a measure of the toxic effect of the chemical.

Use of two short-term tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures.

The genotoxicity of river water samples was evaluated by the Salmonella mutagenicity assay and by the microscreen phage-induction assay. Different processes of sample treatment were compared using the following assays: different volumes of a non-concentrated sample (direct method); concentrated sample fractionated into portions with acid, basic and neutral activity (liquid-liquid extraction method); sample submitted to extraction of volatile substances (volatile extraction method). Samples that were positive to the Salmonella assay by the direct concentration method lost this activity after liquid-liquid extraction. This difference was related to the loss of substances that volatilize during the extraction process. The study of volatile product concentrates confirmed the role of these compounds in inducing activity present in some samples. The microscreen phage-induction assay proved to be a good screening assay for genotoxic compounds present in small concentration in environmental samples. We conclude that, whenever possible, samples should be treated by the direct method in different volumes to prevent the loss of genotoxic substances.

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis of Salmonella typhimurium cells and phages.

The effect of caffeine on nitrosoguanidine-induced mutagenesis of Salmonella typhimurium and its P22 and L phages was studied. The detected mutations included phage "clear" mutations, reversions of phage "amber" mutation, and prototrophic reversions of the his- auxotroph of Salmonella typhimurium. Neither the recA mutation of the host nor the erf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30--60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effect was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of the recA cell gene or the erf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.

Subinhibitory concentrations of organic ammonium salts: the effect on biological properties of Salmonella typhimurium.

The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells. The culture filtrates were prepared by 1-d cultivation of Salmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37 degrees C after addition of subinhibitory concentrations of organic ammonium salts. The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells. The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10(6) PFU/mL. Substances C3, C9 and C12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs. One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substance A16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.

Effects of subinhibitory concentrations of antibiotics on biological properties of Salmonella typhimurium.

We followed the effects of subinhibitory concentrations (sub-MICs) of 7 antibiotics (ticarcilin, cefotaxim, streptomycin, gentamicin, ciprofloxacin, pefloxacin, mitomycin C) on the sensitivity of a Salmonella typhimurium strain to standard bacteriophages, on the phage DNA as well as on the factors of virulence (permeability and cytotoxic activity). The phage type was not changed by the sub-MICs of the tested antibiotics. However, differences were found in culture filtrates prepared from the bacterial suspensions of the strain cultivated with the sub-MICs. Marked inducing effects on phage DNA were exhibited by mitomycin C (1/2, 1/4, 1/8 of the MIC), pefloxacin (1/2, 1/4, 1/8 of the MIC) and ciprofloxacin (1/2, 1/4, weakly also 1/8 of the MIC). Ticarcilin (1/2 of the MIC), like the aminoglycosides streptomycin and gentamicin (1/2, 1/4, 1/8 of the MIC), had a weak effect. Sub-MICs of the studied antibiotics (with the exception of 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of ticarcilin) decreased the permeability reaction in rabbit skin. Most effective was streptomycin (1/2 of the MIC). Sub-MICs of the tested antibiotics (with the exception of 1/4 and 1/8 of the MIC of ciprofloxacin and 1/4 of the MIC of pefloxacin) caused also an inhibition of the factor responsible for morphological changes on Vero cells. Gentamicin and streptomycin were effective at all the sub-MICs tested.

Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties of Salmonella strains.

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied for Salmonella typhimurium and S. enteritidis strains. PAE was induced by 2 x and 4 x MIC of antibiotics studied for 0.5 h. After PAE and PASME their effect on prophage induction of a lysogenic S. typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined. The longest PAE was found after treatment with ciprofloxacin, higher values being observed with S. typhimurium. PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4 x MIC of amikacin with S. enteritidis (6.9h). PASMEs of ciprofloxacin did not allow any regrowth of either strain. For other antibiotics the PASMEs were different while concentrations of 2 x MIC + 0.2 x MIC and 0.3 x MIC, and of 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin did not allow any regrowth of S. enteritidis. PAEs of the antibiotics tested did not affect the Congo red binding by both Salmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenic S. typhimurium strain. We noted the influence of Congo red binding after applying 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin for S. typhimurium and 2 x MIC + 0.1 x MIC for S. enteritidis.

Increasing incidence of resistance to gentamicin and related aminoglycosides in Salmonella typhimurium phage type 204c in England, Wales and Scotland.

Phage type 204c of Salmonella typhimurium (DT 204c) appeared in bovine animals in 1979. It is now the predominant type in cattle in England, Wales and Scotland and ranks in the 10 most common phage types in humans. All strains of DT 204c have been resistant to at least four antimicrobial drugs. In 1979 and 1980 the most common resistance pattern was that of chloramphenicol, streptomycin, sulphonamides, tetracyclines and trimethoprim (CSSuTTm) but since 1981 strains with additional resistance to ampicillin and neomycin-kanamycin (AK) have predominated. Strains resistant to furazolidone (Fu) have caused sporadic outbreaks. Gentamicin resistance (G) appeared in DT 204c in 1983 and gentamicin-resistant strains are increasing in incidence. With the exception of resistance to furazolidone, drug resistance in DT 204c has been plasmid-mediated. Characterisation of gentamicin resistance plasmids in DT 204c of R-type ACGKSSuTTm has demonstrated the existence of three distinct lines, two of which have been found exclusively in cattle and one in cattle and humans. The misguided and often inappropriate use of antimicrobial drugs in calves has contributed to the appearance of multiresistant strains of DT 204c and positive measures to limit range and levels of antimicrobials available to feed manufacturers may be necessary.

Postantibiotic effects of norfloxacin and netilmicin and their influence on the biological properties of Salmonella strains.

The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PA SME) of norfloxacin and netilmicin on two clinical strains--Salmonella typhimurium and Salmonella enteritidis was investigated. After both PAE and PA SME of antibiotics were studied, we determined their effect on the induction of a prophage in the lysogenic S. typhimurium and on Congo red binding by both serovars, as an indicator of invasive ability in vitro. The PAE was induced by 2.MIC and 4.MIC of norfloxacin and netilmicin for 0.5 h. Norfloxacin induced a longer lasting PAE on both Salmonella serovars as compared to netilmicin. Supra-subinhibitory concentrations (PA SMEs) delayed regrowth of tested strains. The PA SMEs of norfloxacin as well as of netilmicin (except 2.MIC + 0.1.MIC concentration) did not allow regrowth of S. enteritidis. The prophage-inductive ability of norfloxacin was more expressive after PA SMEs than PAE. The PA SMEs of netilmicin caused loss of Congo red binding by S. typhimurium cells and decreased this binding by S. enteritidis cells.

Multidrug resistant typhoid fever: therapeutic considerations.

Forty six blood culture positive cases were studied during the current outbreak of multidrug resistant typhoid fever (MRTF). The present outbreak was caused by E1 phage type and organisms were resistant to all commonly used drugs for the treatment of typhoid fever, viz., chloramphenicol (78%), co-trimoxazole (76%) and ampicillin (68%). Treatment failures with chloramphenicol (45.5%) corroborated well with in vitro resistance. No treatment failure was seen with chloramphenicol and ceftriaxone, when these drugs were used in cases infected with sensitive strains. Among the alternative drugs used in cases with in vitro sensitivity, successful clinical response was seen with ceftriaxone (4/4) and cefotaxime (8/9) as compared to cephalexin (3/5) or a combination of cephalexin and furazolidone (9/12).

Characterization of Salmonella enterica serotype typhimurium in the Czech Republic: phage types, antimicrobial and plasmid profiles.

In this study a collection of 547 S. Typhimurium strains isolated in the years 2000 and 2001 both of the human and non-human origin were analysed. 21 different phage types were detected, the most frequent one was DT104 (46%) followed by DT141 (28%) and DT68 (3%). Resistance to one or more antimicrobial agents was found mainly in DT104 (77.4%). S. Typhimurium isolates resistant to 5 and more antimicrobial agents were found in three phagetypes DT104 (57%), DT120 and DT155. Plasmid profiling of DT104 isolates showed 10 different profiles. Pattern A found in 30.5% of tested strains was predominant and carried serovar specific plasmid and one additional small plasmid of approx. 2.5 kb.